Antiviral action of aqueous extracts of propolis from Scaptotrigona aff. postica (Hymenoptera; Apidae) against Zica, Chikungunya, and Mayaro virus

The limited availability of antivirals for new highly pathogenic strains of virus has become a serious public health. Therefore, news products against these pathogens has become an urgent necessity. Among the multiple sources for news antibiotics and antivirals, insect exudates or their products has become an increasingly frequent option. Insects emerged 350 million years ago and have showed a high adaptability and resistance to the most varied biomes. Their survival for so long, in such different environments, is an indication that they have a very efficient protection against environmental infections, despite not having a developed immune system like mammals. Since the ancient civilizations, the products obtained from the bee have been of great pharmacological importance, being used as antimicrobial, anti-inflammatory, antitumor and several other functions. Investigations of biological activity of propolis have been carried out, mainly in the species Apis mellifera, and its product have showed activity against some important viruses. However, for the Meliponini species, known as stingless bees, there are few studies, either on their chemical composition or on their biological activities. The importance of studying these bees is because they come from regions with native forests, and therefore with many species of plants not yet studied, in addition to which they are regions still free of pesticides, which guarantees a greater fidelity of the obtained data. Previous studies by our group with crude hydroalcoholic extract of propolis demonstrated an intense antiviral activity against Herpes, influenza, and rubella viruses. In this work, we chose to use aqueous extracts, which eliminates the presence of other compounds besides those originally present in propolis, in addition to extracting substances different from those obtained in alcoholic extracts. Therefore, this study aimed to identify, isolate and characterize compounds with antiviral effects from aqueous propolis extracts from Scaptotrigona aff postica, in emerging viruses such as zicavirus, chikungunya, and mayaro virus. The evaluation of the antiviral activity of the crude and purified material was performed by reducing infectious foci in VERO cell cultures. The results obtained with crude propolis, indicate a high reduction of zica virus (64×) and mayaro (128×) when was used 10% v/v of propolis. The reduction of chikungunya virus was of 256 fold, even when was used 5% v/v of propolis. The chemical characterization of the compounds present in the extracts was performed by high-pressure liquid chromatography. Through the purification of propolis by HPLC and mass spectrometry, it was possible to identify and isolate a peak with antiviral activity. This substance showed activity against all viruses tested. When purified fraction was used, the reduction observed was of 16 fold for zicavirus, 32 fold for mayaro virus and 512 fold for chikungunya virus. Likewise, it was observed that the antiviral response was concentration dependent, being more intense when propolis was added 2 h after the viral infection. Now we are carrying out the chemical characterization of the purified compounds that showed antiviral action.

resinous material that bees collect from plants, mix with wax, and use to build their nests.Bees apply propolis in thin layers to the inner walls to strengthen and block holes and crevices, as well as use it as an "embalming" substance on dead invaders or even dead bees, thus inhibiting the growth of fungi and bacteria inside the hive 18,19 .The medicinal properties of propolis have been reported since ancient times.In Egypt it was used in the mummification process to prevent bodies from rotting; the Greeks and Romans used it as an antiseptic and healing agent, and the Incas as an antipyretic 19,20 .Due to its great pharmacological potential, propolis became the target of several researches, arousing the interest of the scientific society that has sought to investigate its compounds and biological activities.This interest has intensified in recent decades 18,21 .Countries such as Switzerland and Germany already legally recognize propolis as a medicine 22 .In Brazil, the main substances described are those derived from prenylates of p-coumaric acid and acetophenone, such as diterpenes and lignans 22 .Studies with propolis have already demonstrated biological activities, such as immunomodulation, antitumor, antioxidant, antibacterial, antiviral, and analgesic [23][24][25] .
One of the components of hives is resins.Resins are pre-formed substances produced by secondary metabolites of plants, which have specific functions, such as pigmentation of flowers to attract pollinating insects, repellent action, and defense mechanisms against pathogenic microorganisms 26 .In propolis, the resin is used in prophylactic protection and combat against microorganisms.Studies show that epidemics in nests decrease both productivity 27 and the number of bees 28 .Therefore, the chemical compounds present in these products are essential for the protection and survival of bees, being considered an evolutionary mechanism for the benefit of the colony 29 .The composition of propolis is extremely variable, in terms of both geographic location and season 30 .In Europe, North America, and temperate zones, flavonoid compounds, phenolic acids, and esters predominate in propolis 31 .In the Mediterranean, terpenoids have been identified in high concentrations 32,33 .In Africa, the main compounds are triterpenoids 34 .
In Brazil, due to the great biodiversity, several types of propolis have been reported, such as red, green, and brown.The vast majority of studies are carried out with propolis from bees of commercial interest, mainly Apis mellifera.The propolis of the species Apis mellifera was classified into 12 distinct groups, based on the physicochemical characteristics and place of origin.Among the main compounds isolated are coumaric acid, ferulic acid, pinobanksin, kaempfero, apigenin, and caffeic acid 35 .The components of Brazilian red and green propolis are commonly extracted from Baccharis spp, Dalbergia ecastaphyllum, Betula verrucosa, Betula pendula, and Betula pubescens species and are composed of phenylpropanoids, phenolic acids, p-coumaric acids, diterpenic acids 34 .Studies on the glandular secretions of bees have demonstrated antibacterial activity, as is the case with the hypopharyngeal secretion of the species Apis mellifera 36 .The same hypopharyngeal glands were found in the species Scaptotrigona postica, presuming they have the same functions 37 .The vast majority of studies on the pharmacological effects of propolis are related to propolis obtained from Apis mellifera bees 20 .Not only has the vegetation of the environment but also by the seasons and the bees that produce it 18 influenced the chemical composition of propolis.These variations in chemical components affect their biological properties 38 .For this reason, it is so important to study the propolis of different bee species and regions.Among the different species of bees, studies dealing with propolis from stingless bees are rare 39 .In Brazil, one of the species of stingless bees belongs to the Apidae family, of the Meliponinae subfamily.These bees mix plant resin with wax and clay, thus forming a geopropolis.This denomination is precisely due to the use of land for the final composition of the material produced 39,40 .Recently, compounds such as phenolic acids and water-soluble tannins were isolated from the geopropolis of Melipona fasciculata, (gallotannins and ellagitannins).These compounds, from the aqueous and alcoholic fractions of geopropolis from Melipona scutellaris 41,42 , demonstrated antioxidant activity 43 , antiinflammatory and antinociceptive activities.Within the Meliponinae subfamily, there is the genus Scaptotrigna, which is distributed throughout the Neotropical region and includes species that build their nests in pre-existing cavities 44 .In this genus, we have Scaptotrigona aff postica, popularly known in Maranhão, a region of northeastern Brazil, as the "tubi" bee.However, despite being in the family of geopropolis producers, they are an exception to the rule, since they do not use land in the composition of propolis 21 .
There are few studies with propolis from Scaptotrigona aff postica, and there is no consensus in the literature on how to refer to it, some groups use the name geopropolis 47 , while others use propolis 21,45,46 .For this work, we will use the term propolis, as we understand it to be the most appropriate.This propolis has been used by the population of the Maranhão region in the treatment of cancer and wound healing 21 .Earlier, we have shown that propolis from Scaptotrigona aff postica presents a potent antiviral action against Herpes Simplex Virus (HSV-1) 47 as well against Rubella virus 48 .Scientific studies have corroborated the possible medicinal effects of this propolis.Recently, we have shown 49 that propolis and its components can help reduce the physio pathological consequences of covid-19 infection, and that, flavonoids glycosides and pyrrolizidine alkaloids from propolis of Scaptotrigona aff.postica has a potent antimicrobial action 50 .Martin et al. 51 treated rats with corneal injuries caused by burns with an emulsion of the crude extract and observed both healing and anti-inflammatory effects.Antitumor activity against Ehrlich tumor and reduction in pathology associated with asthma due to an inhibition of migration of inflammatory cells into the alveolar space are also described.In addition to those mentioned, there are no reports on other actions of this product.Therefore, due to the importance of these arboviruses in public health, the limited availability of effective drugs on the market, and the abundant information on the antiviral activity of propolis from several bee species, this work aims to study the antiviral activities of propolis from Scaptotrigona.aff postica against arboviruses.

Preparation of crude extract of propolis
In this work, was used aqueous extraction of propolis as a source of substances for studies of antiviral action.Aqueous extraction of propolis was carried out as described below.The propolis, obtained by scraping the meliponiculture box, was gathered, forming a "paste".This material was transported to the laboratory and frozen at − 20 °C for 24 h, forming a frozen propolis "stone" to facilitate the spraying process.This "stone" of propolis was then manually macerated until it became a very fine granular material.This crushed material was passed through stainless granulometric sieves, fine mesh of 0.15 mm.The sieved material was ground again by one degree until the material above became a very fine powder which was passed again through ABNT (Brazilian Association of Technical Standards) granulometric (75-100 mm Astn) sieves with a porosity of 1-5 microns.After this procedure, ultrapure water was added to the powder (100 ml of water for every 10 g of propolis powders) and homogenized under rotation at 1000 rpm for 24 h at room temperature, protected from light.To separate the precipitate (wax) from the liquid medium (extract), the supernatant was centrifuged at 4 °C for 30 min at 15,000 rpm (Eppendorf centrifuge 5804R).The supernatant was then filtered through a 0.22 µm Millipore membrane, aliquoted, and stored in a sterile glass refrigerator until use.This material was considered to be 100%.

Cell line, culture medium and cell culture
Were used VERO cells (African green monkey kidney fibroblast cells-Cercopithecus aethiops) (Vero-CCL-81, obtained from the ATCC) in this study to determine the antiviral action of propolis.These cells were cultured at 37 °C in T-flasks or 96-well microplates containing Leibovitz medium (L-15), supplemented with 10% fetal bovine serum.Cells were maintained at 37 °C in a cell culture oven.Cell growth and morphology were monitored daily using an inverted optical microscope Olympus CK2.

Cytotoxicity assay of samples in cells
The cytotoxicity test of all samples and the purified fractions of propolis was performed in VERO cells to determine the maximum concentration of propolis or its fractions that is not toxic.For this, cells were seeded at a concentration of 10 4 cells/well in 96-well plates and cultured at 37 °C for 24 h.After this period, the cells were treated with different concentrations of the samples to be tested.PBS was used as a negative control and DMSO 10% as a positive control.After 24, 48, and 72 h of treatment, cell viability was determined by visualizing cell morphology and treating the cells with a vital dye, trypan blue.In each experiment, an aliquot of the samples, at the concentration to be used in the tests, was added to a plate excavation containing VERO cells, as a way of verifying the non-toxicity of the sample, serving as a basis for comparing the morphology of the cells in the test excavations.

Virus
The antiviral action of Scaptotrigona aff postica propolis was tested in three different arbovirus of interest in public health (zica virus, chikungunya, and mayaro vírus).These viruses are part of the collection of the Parasitology laboratory at Butantan Institute.Zika virus (ZIKV, BeH815744) was kindly provided by Dr Pedro Vasconcelos (Evandro Chagas Institute), Mayaro virus (MAYV, BeAr20290) was kindly provided by Dr Maurício Nogueira (São José do Rio Preto School of Medicine), Chikungunya virus (CHIKV) was isolated from a serum sample, which was kindly provided by Dr Alessandra Schanoski (Bacteriology Laboratory, Butantan Institute).

Viral titulation
Initial viral stocks were titrated at the beginning of the work by the viral extinction technique as described by Reed and Muench 52 and detailed in the following item.The viral titer was expressed as TCID50/mL (50% Tissue culture infectious dose), which is the highest dilution of the virus capable of infecting 50% of the cells.To determine the viral titer, aliquots of the virus samples under study were placed on a semi-confluency mat of VERO cells (2 × 10 5 cells/mL), grown in 96 microplates.The viruses were added to the cell cultures in two-fold serial dilutions.The microplates were incubated at 37 °C, the viral cytopathic effect was observed daily, and the titer of infectious viral particles was calculated as described by Reed and Muench.For this study, an aliquot of the virus was thawed immediately before experimenting.

Test of antiviral activity
Test of antiviral activity in cell culture by the technique of determination of the minimum infective dose (TCID/50).In the first phase of tests, VERO cells were treated with crude samples of the test substances, 1 h before infection.After this treatment period, the cells were infected with the different viral strains in serial dilutions ratio 2, with the first dilution varying from 100 to 500 TCI/50 of virus.The cultures were kept at 37 °C and observed daily under an optical microscope, seeking to determine the inhibitory effect of the samples www.nature.com/scientificreports/ on virus replication by determining the cytopathic effect.After the appearance of the cytopathic effect, the culture supernatant was removed and the cells were stained with crystal violet to reveal the cytopathic effect.The antiviral effect was determined by the difference in the highest dilution of the virus capable of inducing a cytopathic effect observed in the infected-only culture and the infected but treated culture.Negative controls for infection and cytotoxicity of the samples were performed in all experiments.
Dose/effect determination.To define the best amount of fraction to be added to infected cultures, fractions showing antiviral activity were added 1 h before infection at concentrations of 2, 5, or 10% (v/v).The cultures were kept at 37 °C and observed daily under an optical microscope, seeking to determine the inhibitory effect of the samples on virus replication through the determination of the cytopathic effect and the determination of the effect performed as described above.
Determination of the optimal time of infection (TOI).To define the best time of infection (TOI), the fractions that showed antiviral activity were tested for the best time to add these fractions to the infected cultures.For this, samples of the fractions, at the optimal concentration described above, were added 2 h before infection, 2 h after infection, or the cultures were treated with a mixture of fraction with virus and kept in contact for 2 h, before being added to crops.The cultures were kept at 37 °C and observed daily under an optical microscope, seeking to determine the inhibitory effect of the samples on virus replication as described above.Six days after the start of the experiment, the supernatant was removed, 0.25% Crystal Violet was added and washed with PBS.

Fractionation of samples by reverse-phase HPLC and ntiviral activity test of purified fractions
Aqueous Propolis Extract HPLC Purification Scapotrigona aff, postica propolis (1,2g) was suspended in 180 mL of ultrapure water and homogenized for 2 h at 4 °C.The supernatant (20 mL) was fractionated by a reverse-phase high-performance liquid chromatography (RP-HPLC) at room temperature on a Shimadzu LC-10 HPLC system using a semi-preparative C18 Jupiter column (30 mm; 300A; 10 LJ 250 mm) (Phenomenex International, Torrance, CA, USA) equilibrated at room temperature with 0.1% TFA in ultrapure water.The elution was done under a linear ACN gradient and the flow rate was 8 mL/min during 60 min (0-80% of B).Fractions with 1 mL was collected, under Ultraviolet absorbance at 225 nm (74).Eluted fractions were immediately refrigerated, concentrated in a vacuum centrifuge SpeedVac Savant (Thermo Fisher Scientific, Waltham, MA, USA), and stored in the freezer at − 20 °C until its usage.The concentrated fractions were reconstituted in 1 mL ultrapure water and used in antiviral activity assays.The purified fractions were tested for their antiviral capacity in the same way as performed with the crude propolis sample.

Characterization of propolis fractions with activity by mass spectrometry
The substances with activity were suspended in 20 μL in ACN/H2O 1:1 with 0.1% formic acid and analyzed by direct infusion in a Thermo Scientific LTQ-Orbitrap LC/MS (LTQ--Linear Trap Quadrupole).

Protein dosage
The concentration of the fractions was determined by the Lambert-Beer law, using the molar extinction coefficient at 205 nm absorption 53 .The active fractions concentration was by measuring A205 nm using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific).

Determination of the cytotoxicity of the agents to be tested
At the beginning of the work, the propolis cytotoxicity of Scaptotrigona aff.postica was determined in VERO cells.The concentrations used were the same used in the tests.For this, VERO cells were grown in 96-well microplates.
When the cell reached confluently, 1, 2, 5, or 10% v/v propolis or its active recombinant analog was added to the cultures in triplicate.The cultures were observed daily and for 72 h to determine the morphological alteration in the cultures.None of the concentrations used showed cytotoxic effects in the cultures.As a negative control of the experiments, in all the tests performed, an aliquot of the agent to be tested was always added to the normal VERO cells, at the same concentration as the test and used for comparison with the results obtained.None of the concentrations tested had a deleterious effect on the growth of Vero cells (Fig. 1).These data suggest that the www.nature.com/scientificreports/propolis studied by us, even in higher concentrations, may be safe for use, which would allow greater antiviral activity to be obtained.Cells were observed under an inverted microscope at 10-x magnification.

Chromatography of the propolis aqueous extract and identification of fractions with viral active
To identify the molecules present in the sample with antiviral action, phytochemical analysis of the propolis extract was performed.The extract was fractionated by a reverse-phase high-performance liquid chromatography (RP-HPLC) at room temperature on a Shimadzu LC-10 HPLC system using a semi-preparative C18 Jupiter column (10 µm; 300 A; 30 mm × 250 mm) (Phenomenex International, Torrance, CA, USA) equilibrated at room temperature with 0.1% TFA in ultrapure water.The elution was done under a linear ACN gradient and the flow rate was 8 mL/min during 60 min (0-80% of B) (Fig. 2A).Each fraction (1 mL) was collected, under Ultraviolet (74) absorbance monitored at 225 nm.Eluted fractions were immediately refrigerated, concentrated in a vacuum centrifuge SpeedVac Savant (Thermo Fisher Scientific, Waltham, MA, USA), and stored in the freezer www.nature.com/scientificreports/ at − 20 °C until its usage.The concentrated fractions were reconstituted in 1 mL ultrapure water and used in antiviral activity assays.The fractions were tested for their antiviral capacity in the same way as performed with the crude propolis sample.Fraction 1 was the one that showed antiviral activity.The fraction 1 was then subjected to mass spectrometry using a Thermo Scientific LTQ-Orbitrap LC/MS (LTQ-Linear Trap Quadrupole).As can be observed in Fig. 2B, various ions were obtained.These ions were submitted to MagTran for mass analysis and identification (deconvolution), and revealed a molecule of mass of 1331.96Da and a concentration of 107 µg/Ml.

Determination of the antiviral action of propolis on different arboviruses
In this work, the antiviral action of propolis was tested against zica virus, chikungunya, and mayaro virus.For this experiment, plates from 96 wells with VERO cells were treated with propolis being maintained in contact with the cells for 1 h.After this period, serial dilutions (ratio 2) of the virus, with an initial titer of 100-500 TCID/50, were added to the treated cultures and observed for 72 h to determine the cytopathic effect.
Antiviral action of propolis and its purified fraction against Zica virus.The test was performed as described above.As can be seen in Fig. 3, there is an intense reduction in the viral of zica virus replication against 256 TCID/50 of virus after the treatment of cells with 10% v/v propolis.Under these conditions, the viral reduction ranged from 256 to 4 TCID/50, or 64 fold (98%) with crude propolis.When cells were treated with purified fraction 1, the antiviral action was of 16-fold (256 to 16 TCID/50).
In the photos in Fig. 4, can be seen that there was a clear reduction in the cytopathic effect (B) caused by the zica virus 72 h after infection, in cultures treated with propolis (10% v/v) (C), which presents a normal morphological aspect as control cells (A).www.nature.com/scientificreports/Antiviral action of propolis against mayaro virus.The antiviral action of the aqueous extract of propolis was also tested against the mayaro virus.The experimental procedures were the same as mentioned above to zica virus, but using 512 TCID/50 of virus.Under these conditions, the viral reduction was similar to that obtained in the tests with the zica virus, as with crude or as purified propolis fraction.In this case, the reduction was on average 128-fold for crude propolis (512 to 4 TCID50) and 32-fold for the fraction (512 to 16 TCID/50) (Fig. 5).

The result represents four individual experiments (performed in duplicate each).
As ocurred with zica virus, a clear reduction in the cytopathic effect caused by the mayaro virus 72 h after infection, in cultures treated with propolis (10% v/v).(Fig. 6).
Antiviral action of propolis on chikungunya virus.The antiviral action of the aqueous extract of propolis was tested against chikungunya virus.The experimental procedures were the same as mentioned above, only with a greater amount of virus (1000 TCID/50%) and 5% propolis.As can be seen in Fig. 7, a 256-fold reduction in viral replication was observed when crude propolis was added.When the purified fraction was used the virus reduction was of 512-fold.
In the photos in Fig. 8, it can be seen that there was a clear reduction in the cytopathic effect of chikungunya virus, when 5% (v/v) of propolis or its purified fractions was used.

Effect of the concentration of propolis aqueous extract on viral replication
The action of concentration of propolis aqueous extract on antiviral activity was tested with zica virus or with mayaro.For this, the VERO cell cultures were treated with 2, 5, or 10% propolis and after 1 h the cells were infected with 128 or 512 TCID/50% of zica virus or mayaro virus.The cultures were observed for 72 h and the viral titer was defined as the highest dilution of the virus capable of inducing a cytopathic effect in the cells.As can Figure 5. Antiviral action of propolis from Scaptotrigona aff.postica against the Mayaro virus.Aqueous propolis extract (10% v/v) was added to the VERO cell culture.One hour after this procedure, serial dilutions (r:2) of the Mayaro virus (512 TCID 50/0.1 ml) were added to the culture.The culture was maintained at 37 °C for 72 h and the culture was observed daily to determine the cytopathic effect.The final titer was determined by the highest dilution of the virus to cause a cytopathic effect in the culture.

Figure 6. Photomicrography of normal VERO cell cultures (A), infected cells with Mayaro virus (B)
, and infected cells treated with 10% v/v propolis from Scaptotrigona aff.postica (C).The cultures were kept for 3 days at 37 °C.Cells were observed daily under an inverted microscope at 10 × magnification.After this period, the culture supernatant was removed and the cells were stained with crystal violet 025%.be seen in Fig. 9, the antiviral action of propolis is dose-dependent, and the viral reduction with the concentration of 10% of propolis extract was 64-fold to zica virus and 128-fold to the mayaro virus (99.6% of reduction).

Effect of time of addition of aqueous propolis extract on viral replication
The effect of the time of addition of the aqueous extract of propolis on the antiviral activity was tested against different dilutions of zica virus (from zero to 2048 TCID/50/0.1 ml).For this, VERO cell cultures were treated with 5% propolis 2 h before infection, 2 h after infection, or treated with a mixture of virus + propolis kept together for 2 h.The culture was observed for 6 days and the viral titer was defined as the highest dilution of the   Zicavirus dilutions (from 0 to 2048 TCID/50/0.1 ml).For this, VERO cell cultures were treated with 5% propolis 2 h before infection, 2 h after infection, or treated with a mixture of virus + propolis kept together for 2 h.The culture was observed for 6 days and the viral titer was defined as the highest dilution of the virus capable of inducing a cytopathic effect in the cells.www.nature.com/scientificreports/virus capable of inducing a cytopathic effect in the cells.As can be seen in Fig. 10, the antiviral action of propolis was more effective when propolis was added 2 h after infection.The experiments were performed in triplicate.

Discussion
Arboviruses are viruses transmitted by arthropods (Arthropod-borne virus) and are so named because part of their replicative cycle occurs in insects.They are transmitted to humans and other animals by the bite of bloodsucking arthropods (mosquitoes).Of the more than 545 known arbovirus species, about 150 cause disease in humans 4 .Arboviruses have represented a major challenge to public health, due to climate and environmental changes and deforestation that favor amplification, and viral transmission, in addition to the transposition of the barrier between species.Among the arboviruses that cause diseases in humans and other warm-blooded animals are Dengue, Zica, Chikungunya, and Mayaro, among many others.These viruses are a major problem, for which there is no specific treatment.Treatment to these viruses is usually focused only on relieving symptoms because there are no drugs that specifically attack the virus 54 .In general, medications are indicated to control fever, headache, skin spots, and body pain, the same used in patients with dengue, zika virus, or other arboviruses.Therefore is extremely important the development of drugs that acts directly on the virus and not just on the symptoms.Various studies have reported the antiviral activity in products obtained from invertebrates and their products.Among the various animal sources of antiviral studies, propolis has gained great attention for obtaining new drugs 16,55 , mainly because of its extensive and varied chemical composition 16,30,56 that generate huge amounts of molecules of high pharmacological value.Peter et al. 57 , describe the antiviral effect of three hydroalcoholic extracts of propolis (brown, green, and jataí bees (Tetragonisca angustula), against bovine herpesvirus type-1 (BoHV-1) and bovine viral diarrhea Virus (BVDV).This propolis has shown high efficacy in both cellular treatments (post and pre-exposure) against BoHV1.Extracts from Jataí showed activity agains46, t both viruses (BoHV-1 and BVDV) infection in the pre-test, whereas brown propolis demonstrated action only against the BoHV-1 in the pre-infection method., being that, the green propolis, led to a reduction in viral titer of 4.33log, while was observed a reduction of 3.5log to brown propolis and of the 3.24log to jataí propolis.Like this one, most of the scientific work on the therapeutic activities of propolis has been carried out with the bee species Apis mellifera.However, some studies using propolis from stingless bees have been described, demonstrating its therapeutic potential against viruses of importance in human and veterinary health such as human herpes virus type 1 47,58,59 , Herpes Vírus Bovino (BoHV-5) 60 , Parvovírus Suíno (PPV) 61 , vírus da imunodeficiência human (HIV) 62 and influenza virus 63 .
One of the advantages of propolis as a therapeutic agent is, in general, the absence of toxicity to humans and animals 15 .Studies have been performed in which the concentrations of propolis used in antiviral assays vary from 0.0003 to 5% 58 , with no morphological alterations being observed in the cells 15 .Peter et al. 57 , observed cytotoxicity wih 0.097 µg/mL for brown and green propolis and 0.39 µg/mL for jataí propolis.In a similar study, Bankova et al. 16 evaluated the antiviral activity of a hydroalcoholic extract of Canadian propolis, where it was observed that the maximum tolerated non-cytotoxic concentration of propolis in MDBK cells was 0.032 mg/ mL.This relative toxicity may be related to the extraction method used, which was a hydroalcoholic extraction, which removes a good amount of bioactive substances present in propolis.In our experiments were carried out aqueous extraction.With this procedure, substances that are not isolated in alcoholic extracts are obtained, which leads to the discovery of new molecules with therapeutic potential.This procedure also avoids the presence of other substances that may be toxic.In our study, with the aqueous extraction, we did not observe any toxic effect on VERO cells, when we used final concentrations of up to 10% of propolis, which allows for obtaining a higher concentration of bioactive products in the formulation.
There are several reports of the antiviral activity of propolis.However, the mechanisms of action of propolis on viruses are not yet fully elucidated, but some studies seek to identify where this activity occurs.Bankova et al. 16 evaluated an alcoholic extract of propolis against Herpes simplex type 2 (HSV2) before contact with the cell, and suggest that the action of propolis occurs in the structure of the viral envelope or by modifying structural components necessary for adsorption or entry of the virus into the cell.The same study was carried out against Herpes simplex type 1 (HSV1), and the data suggest that when propolis is added before or at the time of infection, a greater virus-inhibiting effect is observed.In the study by Peter et al. 57 with extracts of brown propolis and jataí, a greater effect of propolis was observed when previously inoculated the cells, before viral infection, suggesting the destruction of the virus inside the cell.However, the same result was not observed when this propolis sample was inoculated previously to BVDV, possibly showing different mechanisms of viral neutralization.There appears to be a wide variation in the activity of different types of propolis in antiviral activity.When Peter et al. 57 used green propolis extract, they did not observe significant antiviral activity in any of the experiments used, which was contrary to what was observed with brown and Jataí propolis, These data with green propolis differ from those obtained by Fischer et al. 60 , who evaluated the effect of the antiviral activity of a Brazilian green propolis sample against bovine herpesvirus type 1 and BVDV.These differences may be related to the chemical composition of propolis from different origin.Recently, in a study carried out by us with 12-month samples of propolis from Scaptotrigona aff.postica, we have observed a great variation in the chemical composition of propolis according to the harvest month.This variation in chemical composition is related to the variation of flowering plants in each month, which bees use for the production of honey and propolis.Unlike other studies carried out with propolis from other sources, which derives exclusively or mostly from a single botanical source, the propolis from Barra do Corda used in our studies is composed of several sources, as it was obtained in a region of native forest, with a very large variety of plants 30 .
One of the positive aspects of this propolis is that its origin is in a native region, without the use of pesticides, which prevents its contamination by other unnatural products.Cueto et al. 64  www.nature.com/scientificreports/feline Calicivirus, Canine Adenovirus type 2, and Bovine Viral Diarrhea Virus.In this study, the presence of flavonoids such as rutin, quercetin, and gallic acid was identified.In this work, antiviral activity against BVDV and CAV-2 was observed when propolis was added before viral infection.In a study carried out by us 47 , we determined the antiviral action of a hydroethanolic extract of Scaptotrigona aff.postica from the region of Barra de Corda, state of Maranhão, Brazil against Herpes Simplex Virus (HSV-1).The chemical analysis of propolis showed higher amounts of pyrrolizidine alkaloids and C-glycosyl flavones, which is a compound detected for the first time in a sample of origin of meliponian bees.Apparently, the viral type also influences the results of antiviral activity.Peter et al. 57 , observed that jataí propolis -T.angustula) obtained better antiviral results against RNA virus (BVDV) than when using DNA virus (BoHV-1).The BVDV genome consists of a single strand of RNA, surrounded by a lipid envelope, which constitutes a structural difference concerning BoHV-1, which is a DNA virus 64 .Eventually, this may explain the greater action of the propolis obtained from the jataí bee.Yildirim et al. 59 observed a significant reduction in HSV-1 and HSV-2 replication with Jatay propolis.However, toxic action was observed at concentrations of 200 and 400 μg/mL.The time of action of propolis also varied according to the virus used.While the replication of HSV-1 started 24 h after infection, for HSV-2 the start of replication was after 48 h of incubation.In our study, propolis of Scaptotrigona aff postica was tested, for potential antiviral activity against arboviruses.Experiments with total propolis led to an average 256-fold reduction in replication of the three tested viruses, while the semi-purified protein led to a 512-fold reduction in chikungunya virus production, a 64-fold reduction in mayaro replication, and a 16-fold reduction in Zica virus production.The action of propolis was higher when the cell culture was infected 2 h before the addition of propolis, which suggests an intracellular mechanism of action.In this case, the substance present in propolis, may act as a constitutive agent that affects the innate antiviral immune response.IFNs, cytokines and chemokines are seen as essential for efficient host defense against infection.Melchjorsen et al. 65 showed that the initial response of HSVinfected cells is the secretion of antiviral substances such as defensins and nitric oxide and cytokine production, including IFN and chemokines.Earlier we also reported that type I IFNs stimulate Nitric Oxide Production and Resistance to Trypanosoma cruzi Infection 66 .As occur with trypanosomes, we believe that the mechanism of action of some antivirals is to stimulate cells to produce interferon, nitric oxide, or other cytokines.On the other hand, when the virus was kept in contact with propolis, although there was a good effect, it was not as intense as when propolis was added before, which even suggests an intracellular action.The same occurred when the virus was added before of propolis addition, which suggests that the effect was not due to the action of propolis on the membrane.Nevertheless, its mechanism of action needs further investigation.Concluding, this study showed antiviral activity against various arbovirus.It is can be an important target for the development of new compounds as an alternative to commercial antivirals.In addition to the antiviral activity, it is expected to obtain many other pharmacological activities in the propolis of Scaptotrigona aff.postica, given the great complexity in the composition of this product and the great variability in the seasonal chemical composition that we observed in this propolis 30 .

Figure 1 .
Figure 1.Cell viability of Vero cells treated with differents concentrations of propolis.The culture were treated com 1,2,5 or 10% v/v of propolis.The cultures were maintained at 37º C for 72 h and the cultures were observed daily to determine the cytotoxic effect.The results above represents the morphology observed in cels in all experiments.

Figure 2 .
Figure 2. Phytochemical analysis of the propolis aqueous extract.(A) Reverse-phase high-performance liquid chromatography (RP-HPLC): samples eluted with a linear gradient from 0 to 80% ACN in acidified water for 60 min (8 ml/min.).The fractions were collected manually and submitted to a test of the inhibition of microbial growth in a liquid medium.(B) The fraction 1 (with antiviral activity) was resuspended in 20 μL in ACN/H2O 1:1 with 0.1% formic acid and analyzed by direct infusion in a Thermo Scientific LTQ-Orbitrap LC/MS (LTQ-Linear Trap Quadrupole), under 0.5 μL/min).Mass spectrometry analyses revealed some ions (m/z) the most abundant being an interferent (371 m/z).Ions were submitted to MagTran for mass analysis and identification (deconvolution), and revealed a molecule of mass of 1,331.96Da.

Figure 3 .
Figure 3. Antiviral action of propolis from Scaptotrigona aff.postica (10% v/v) against the Zica virus.The propolis extract or the purified fractions were added to the cultures of VERO cells and 1 h later, serial dilutions (r:2) of Zica virus (initial titer of 256 TCID 50/0.1 ml) were added to the culture.The culture was maintained at 37 ºC for 72 h and the culture was observed daily to determine the cytopathic effect.The final titer was determined by the highest dilution of the virus to cause a cytopathic effect in the culture.The result represents 4 individual experiments (performed in duplicate each.

Figure 4 .
Figure 4. Photomicrography of normal VERO cell cultures (A), infected with Zica virus (B) and infected and treated with 10% v/v propolis from Scaptotrigona aff.postica (C).The cultures were kept for 3 days at 37 °C.Cells were observed daily under an inverted microscope at 10 × magnification.After this period, the culture supernatant was removed and the cells stained with crystal violet (0,25%).

Figure 7 .
Figure 7. Antiviral activity of the aqueous extract of propolis from Scaptotrigona aff.Postica (5% v/v) against tne Chikungunya virus.The propolis extract or the purified fractions were added to the Vero cell cultures.1 h later, serial dilutions (r:2) of the virus, with initial titer of 1000 TCDI 50/0.1 ml, were added to the culture.The culture were maintained at 37 °C for 72 hous and the cultures were observed daily to determine the cytophatic effect.The final titer were determined by the higtest dilution of the virus to cause a cytohatic effect in the culture.The date in the figure are representative of 4 individual experiments, performed in duplicate each one.

Figure 8 . 7 Figure 9 .
Figure 8. Photomicrography of normal VERO cell cultures (A), infected with Chikungunya virus (B), and infected cells treated with 5% v/v propolis from Scaptotrigona aff.postica (C) or with infected and treated with 5% of purified fraction of propolis (D).The cultures were kept for 3 days at 37 °C.Cells were observed daily under an inverted microscope at 10 × magnification (A, B, and C) and 20 × in photo D. After this period, the culture supernatant was removed and the cells stained with crystal violet 025%.

Figure 10 .
Figure10.Action of propolis aqueous extract (TOI) addition time on antiviral activity against different Zicavirus dilutions (from 0 to 2048 TCID/50/0.1 ml).For this, VERO cell cultures were treated with 5% propolis 2 h before infection, 2 h after infection, or treated with a mixture of virus + propolis kept together for 2 h.The culture was observed for 6 days and the viral titer was defined as the highest dilution of the virus capable of inducing a cytopathic effect in the cells.

Material and methods Propolis The
) propolis used in this work was obtained from a colony of Scaptotrigona aff postica, located in the State of Maranhão, in the Barra do Corda region, Brazil from apiary Melinative.The city is located in the geographic center of Maranhão, at the confluence of the Rio da Corda and Rio Mearim.The propolis used in this work was obtained from a colony of Scaptotrigona aff postica, located in the State of Maranhão, in the Barra do Corda region, Brazil from apiary Melinative.The city is located in the geographic center of Maranhão, at the confluence of the Rio da Corda and Rio Mearim.